Bcl-2 family proteins are known to regulate a distal step in an evolutionarily conserved pathway for programmed cell death, with some members functioning as suppressors of apoptosis and others as promoters of cell death (Gross, et al., Genes Dev. 13:1899–1911, 1999; Reed, Nature 387:773–776, 1997). In mammalian cells, Bcl-2 family proteins are known to control mitochondria-dependent cell death cascades (Adams and Cory, Science 281:1322–1326, 1998; Green and Reed, Science 281:1309–1312, 1998; Reed, et al., Cancer J. Sci. Am. 4 Suppl. 1:S8–14, 1998). Mitochondria release apoptogenic factors during apoptosis such as cytochrome c apoptosis-inducing factor (AIF), and SMAC/DIABLO (Green, 2000). cytochrome c released from mitochondria into the cytosol space triggers Apaf-1-dependent caspase activation leading cells to death (Green, Cell 102:1–4, 2000; Zou, et al., Cell 90:405–413, 1997). Pro-apoptotic Bcl-2 family proteins such as Bax promote cytochrome c release from mitochondria (Jurgensmeier, et al., Proc. Natl. Acad. Sci. USA 95:4997–5002, 1998). On the other hand, anti-apoptotic Bcl-2 family proteins such as Bcl-2 suppress cytochrome c release from mitochondria, thereby protecting cells from apoptotic signals triggered by several stimuli (Kluck, et al., Science 275:1132–1136, 1997; Yang, et al., Science 275:1129–1132, 1997). The relative ratios of these various pro- and anti-apoptotic members of the Bcl-2 family have been known to determine the sensitivity of cells to diverse apoptotic stimuli (Oltvai and Korsmeyer, Cell 79:189–192, 1994) including chemotherapeutic drugs and radiation, growth factor deprivation, loss of cell attachment to extracellular matrix, hypoxia (a common occurrence in the centers of large tumors), and lysis by cytotoxic T-cells (Adams and Cory, supra, 1998; Green and Reed, supra, 1998; Gross, et al., supra, 1999; Reed, Semin. Hematol. 34:9–19, 1997).
Among pro-apoptotic Bcl-2 family members, Bax and Bak play a key role for apoptosis induction. The double knock out of these genes in mice resulted in the resistance of the cells to several cell death stimuli known to trigger mitochondria-dependent apoptosis, such as UV-irradiation, staurosporin (pan-kinase inhibitor), and some anti-cancer drugs (Wei, et al., Science 292:727–730, 2001). Bax normally resides in the cytosol in a quiescent state. Upon receipt of apoptotic stimuli, Bax translocates into mitochondria (Wolter, et al., J. Cell. Biol. 139:1281–1292, 1997), and promotes cytochrome c release, possibly by forming a pore in the mitochondrial outer membrane (Korsmeyer, et al., Cell Death Differ. 7:1166–1173, 2000; Saito, et al., Nat. Cell Biol. 2:553–555, 2000). On the other hand, anti-apoptotic family proteins such as Bcl-2 and Bcl-XL reside in the mitochondrial membrane and antagonize the cytotoxic activity of Bax moved from the cytosol (Adams and Cory, supra, 1998; Green and Reed, supra, 1998; Reed, et al., supra, 1998). Mitochondrial translocation of Bax is one of the critical steps for the induction of apoptosis, however the mechanism is not yet fully understood.
Translocation of Bax from the cytosol to the mitochondria is caspase-independent, since caspase-inhibitor pretreatment does not interfere with this process (Goping, et al., J. Cell Biol. 143:207–215, 1998). C-terminus hydrophobic residues forming the ninth (α-helix of Bax are reported to be involved in the translocation of Bax to the mitochondrial membrane (Suzuki, et al., Cell 103:645–654, 2000). On the other hand, the N-terminus of Bax functions as a cytosol retention domain, since the deletion of this region allowed Bax to accumulate in the mitochondrial membrane in the absence of apoptotic stimuli (Goping, et al., supra, 1998). The previous observations suggest that unidentified cytosolic factor(s) interact with the N-terminus of Bax to inhibit its translocation to mitochondria in the absence of an apoptotic stimulus.
U.S. application Ser. No. 10/247,045 describes the suppression of BAX by Ku-70, a factor that binds the N-terminus of Bax and prevents its mitochondrial translocation. The present invention involves the development of a membrane permeable peptide that inhibits Bax-mediated apoptosis.